Pathological changes in muscle stiffness can result from disease, blunt trauma, overuse, or as secondary complications from other injuries and treatments. Changes in muscle stiffness can lead to reduced mobility, chronic pain, discoordination, and increased rate of injury. Consequently, many therapeutic interventions target muscle or joint stiffness. While there is a long history of measuring joint stiffness, there are no validated methods to directly quantify the intrinsic stiffness of individual muscles independently from the other factors influencing the mechanics of a joint. This is a major obstacle to identifying, treating, and monitoring muscle contributions to stiffness-related impairments. Our long-term goal is to improve treatments for musculoskeletal disorders associated with changes to the intrinsic properties of muscle. The central hypothesis of this proposal is that ultrasound elastography (USE), a relatively new imaging tool for the clinic, can be used to measure the intrinsic stiffness of living muscles. Variants of this hypothesis have been widely assumed, but not directly tested aside from the preliminary data provided in this proposal. Our rationale is that providing an objective measure of intrinsic muscle stiffness will clarify the role of muscle in stiffness-related impairments, and lead to a personalized approach to treatment design and evaluation. Our central hypothesis will be evaluated using three aims. Aim 1 will determine the extent to which elastography can measure the intrinsic stiffness of muscles during active contractions. This will be completed in architecturally different muscles of the cat hind limb, where activation can be controlled precisely and direct mechanical measures obtained for comparison. Parallel human experiments will assess feasibility in clinically relevant settings, when muscle is activated by normal patterns of recruitment and rate modulation. Aim 2 will determine if substantial passive forces alter the stiffness estimates obtained by USE. For clinical utility, USE must provide accurate measures during the many conditions in which passive and active structures are relevant. These experiments will be conducted only in cats, as direct measures of passive muscle force are difficult to obtain in humans. Aim 3 will determine if USE can detect microstructural changes in muscle, which are typically only accessible by invasive techniques such as biopsies. Our prior work demonstrated that magnetic resonance elastography (MRE) is sensitive to changes in tissue structure at scales well below image resolution. This project will determine if USE, a more clinically viable technique, can have a similar sensitivity. This will be evaluated by applying MRE and USE to imaging phantoms created using our ability to print biomaterials with known properties in 3D, and to living muscles from cats and humans. This third aim will extend current technologies to characterize muscle and its underlying microstructure more completely. Together, our results could profoundly impact the way disease-related changes in muscle stiffness are quantified and lead to more targeted interventions that alleviate impairments associated with changes to intrinsic muscle stiffness.